Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two-hybrid system.

The TPD52 (tumor protein D52)-like proteins are small coiled-coil motif-bearing proteins which were first identified though their expression in human breast carcinoma. TPD52-like proteins are known to interact in hetero-and homomeric fashions, but there are no known heterologous binding partners for these proteins. We now report the cloning of a novel member of the MAL proteolipid family, named MAL2, though its interaction with a TPD52L2 bait in a yeast two-hybrid screen. MAL2 is predicted to be 176 residues (19 kDa) with four transmembrane domains and is 35.8% identical to MAL, a proteolipid required in apical vesicle transport. The MAL2 prey bound all TPD52-like baits tested in the yeast two-hybrid system and in vitro translation of MAL2 produced a single 19-kDa (35)S-labeled protein which specifically bound full-length GST-Tpd52 in GST pull-down assays. The gene MAL2, which was localized to human chromosomal band 8q23 and shown to consist of four exons, is predominantly expressed in human kidney, lung, and liver. Our study has therefore identified a novel member of the MAL proteolipid family and potentially implicates TPD52-like proteins in vesicle transport.

Cloning of a third member of the D52 gene family indicates alternative coding sequence usage in D52-like transcripts.

D52 proteins are emerging as signalling molecules which may be regulators of cell proliferation. Having previously reported the existence of the human D52 gene family, comprising the hD52 and hD53 genes expressed in human breast carcinoma, we report the identification of a novel human gene hD54 (TPD52L2), which represents a third D52 gene family member. In situ mapping placed the hD54 gene on human chromosome 20q13.2-q13.3, a localization distinct from those of both hD52 and hD53 genes. The identified hD54 cDNAs predicted three hD54 isoforms, suggesting that alternatively-spliced transcripts may be produced from D52-like genes. This was confirmed by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products amplified from D52-like gene transcripts expressed in developing and adult rat tissues, and by performing sequence analyses of the expressed sequence tag divisions of nucleotide databases. Alternative splicing of sequences encoding two regions, termed ins2 and ins3, was identified in one or more D52-like genes, with these alternative splicing events being differentially regulated. The functional consequences of alternative splicing were examined by characterizing the protein-protein interactions mediated by a truncated hD53 isoform within the yeast two-hybrid system. This hD53 isoform displayed altered interaction capabilities with respect to those of full-length hD53, suggesting that alternative splicing within the D52 gene family functions in part to alter the protein-protein interaction capabilities of encoded isoforms.

Identification of homo- and heteromeric interactions between members of the breast carcinoma-associated D52 protein family using the yeast two-hybrid system.

The hD52 gene was originally identified through its elevated expression level in human breast carcinoma. Cloning of D52 homologues from other species has indicated that D52 may play roles in calcium-mediated signal transduction and cell proliferation. Two human homologues of hD52, hD53 and hD54, have also been identified, demonstrating the existence of a novel gene/protein family. Since D52-like protein sequences are all predicted to contain a coiled-coil domain, we used the yeast two-hybrid system and glutathione S-transferase pull-down assays to investigate whether homo- and/or heteromeric interactions occur between D52-like proteins. Analyses of yeast strains co-transfected with paired D52-like constructs indicated that D52-like fusion proteins interact in homo- and heteromeric fashions through their predicted coiled-coil domains. Similarly, extensive two-hybrid screenings of a human breast carcinoma expression library identified hD53 and hD52 as potential interactors for both hD52 and hD53 baits. Thus, D52-like proteins appear to exert and/or regulate their activities through specific interactions with other D52-like proteins, which in turn may be intrinsic to potential roles of these molecules in controlling cell proliferation.